CCR5 is an HIV-1 coreceptor and mediates viral entry into target cells. Small molecules SCH-C, TAK-779, and the anti-CCR5 MAb PA14 inhibit HIV entry by disrupting gp120-CCR5 binding. IN particular these inhibitors affect the conformation of the second extracellular loop (ECL2) of CCR5. We have characterized 3 isolates which differ by over an order of magnitude in their sensitivity to CCR5 inhibitors. We hypothesize that the crown of the third variable loop (v3) of HIV envelope glycoprotein gp120 interacts with ECL2 and thereby determines SCH-C, TAK-779, & PA14 sensitivity. To test our hypothesis we have generated chimeric gp120 at the level of the V3 crown and stem. These chimeras will be tested for altered sensitivity to CCR5-based inhibitors. The V3 loop may regulate inhibitor sensitivity by modulating gp120 affinity for CCR5 and/or the kinetics of HIV-1 fusion and entry. Using gp120-CCR5 binding assays as well as time course of inhibition experiments, we will determine the precise mechanism of CCR5 inhibitor sensitivity. Overall, better understanding of CCR5 inhibitors will enable us to further modify and perfect these compounds.